Long lasting agonists and antagonists of LH-RH

ABSTRACT

Novel peptides of the formula: ##STR1## where A through G, X and Y are various amino acids and substituents; having long lasting LHRH agonist and antagonist activity; useful in promoting fertility, reducing fertility, respectively.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is concerned with novel peptide compounds havinglong lasting LHRH agonist and antagonist activity, and withpharmaceutical compositions containing said novel peptide compounds, aswell as their use in methods of promoting and methods of reducingfertility.

2. Brief Description of the Prior Art

Luteinizing hormone-releasing hormone (LHRH) is a neurohumoral hormoneproduced in the hypothalamus which stimulates the secretion of thepituitary hormones, luteinizing hormone (LH) and follicle-stimulatinghormone (FSH), which in turn produce changes resulting in the inductionof ovulation. LHRH has the following structure: ##STR2##

Synthetic replicates of LHRH were readily available shortly after theprimary sequence was disclosed in 1971 and, as a result, a sizablenumber of structural analogs of LHRH have been made and tested over thelast few years. Some of these have proved to be more potent than LHRH aswell as long-acting. See Rivier et al., Peptides 1976 (Proceedings ofthe Fourteenth European Peptide Symposium, Wepion, Belgium, Apr. 11-17,1976), pp. 427-436. Thus, a D-amino acid has been utilized in the6-position and/or N-methyl-Leu⁷ substitution has been made in order toobtain potent, long-acting LHRH agonists and antagonists. These priorstudies have also indicated the importance of the backbone conformationto full potency of the analogs.

The novel peptides of the present invention, in contrast to the priorart, possess a five- or six-membered lactam bridge between the 6- and7-positions of the peptide. It is theorized that this novel structureimproves the biologically active (receptor bound) conformation of thepeptide, thus affording greater potency, as well as the resistance ofthe peptide to enzyme degradation, thus extending the useful life of thepeptide.

The novel peptides of the present invention are prepared in accordancewith a novel process. Cyclizations of methionine sulfonium salts whichhave been previously reported have resulted in O-, rather thanN-alkylation. See Yeung, et al., Biochemistry, Vol. 16, p. 1635 (1977).

SUMMARY OF THE INVENTION

The present invention concerns novel peptides of the formula ##STR3##wherein, unless otherwise indicated, an amino or imino acid has the "L-"stereoconfiguration:

A is L- or D-pyroGlu (pyroglutamic acid residue); L- or D- N-acetyl orN-pyroGlu imino acid; acetyl D-Phe (Phenylalanine); or C₃₋₇ cycloalkylacyl;

B is His (Histidine); Gly(Glycine); L- or D- aromatic or aliphatic aminoacid; or is absent;

C is L- or D- aromatic or aliphatic amino acid;

D is Ser (Serine); Thr (Threonine); or Ala (Alanine);

E is aromatic amino acid;

F is amino acid with a basic side chain;

G is imino acid or aliphatic amino acid;

X is GlyNH₂ (Glycineamido); AlaNH₂ (Alanineamido); aminoethyl;aminopropyl; or aminohydroxyethyl;

Y is hydrogen or C₁₋₄ alkyl; and

n is 2 or 3.

Preferred novel agonist peptides of the present invention are:

PyroGlu-His-Trp-Ser-Tyr-6,7-[2-(S-3-amino-2-oxo-pyrrolidin-1-yl)-S-2-isopropylmethylaceticacid]-Arg-Pro-GlyNH₂PyroGlu-His-Trp-Ser-Tyr-6,7-[2-(S-3-amino-2-oxo-pyrrolidin-1-yl)-S-2-isopropylmethylaceticacid]-Arg-Proethylamide

Preferred novel antagonist peptides of the present invention are:

PyroGlu-D-Phe-Trp-Ser-Tyr-6,7-[2-(S-3-amino-2-oxo-pyrrolidin-1-yl)-S-2-isopropylmethaceticacid]-Arg-Pro-GlyNH₂

N-acetyl-Pro-D-Phe-D-Trp-Ser-Tyr-6,7-[2-(S-3-amino-2-oxo-pyrrolidin-1-yl)-S-2-isopropylmethylaceticacid]-Arg-Pro-GlyNH₂

For agonist activity, the A substituent is the same as that forendogenous LHRH, that is, pyroGlu (pyroglutamic acid residue). Forantagonist activity, the A substitutent is L- or D- pyroGlu; L- or D-N-acetyl imino acid, preferably N-acetyl Pro (Proline) or N-acetyl Hyp(Hydroxyproline); L- or D- N-pyroGlu imino acid, preferablyN-pyro-Glu-Pro or N-pyroGlu-Hyp; acetyl D-Phe; or C₃₋₇ cycloalkyl acyl,for example cyclopropylcarbonyl.

For agonist activity, the B substituent is the same as that forendogenous LHRH, that is, His. For antagonist activity, the Bsubstituent is Gly; an L- or D-aromatic amino acid, preferably a memberselected from the group consisting of Phe, Trp (Tryptophan), Tyr(Tyrosine, and His; an L- or D- aliphatic amino acid, preferably amember selected from the group consisting of Ala, Leu (Leucine), Ile(Isoleucine), and Val (Valine); or is absent altogether.

For agonist activity, the C substituent is an aromatic amino acid,preferably a member selected from the group consisting of Phe, Trp, Tyr,and His. For antagonist activity, the C substituent is an L- orD-aromatic or aliphatic amino acid, as described above for substituentB.

For both agonist and antagonist activity, the D substituent is Ser, Thr,or Ala.

For both agonist and antagonist activity, the E substituent is anaromatic amino acid, as described above for substituent C.

For both agonist and antagonist activity, the F substituent is an aminoacid with a basic side chain, preferably a member selected from thegroup consisting of Lys (Lysine), Arg (Arginine), and Orn (Ornithine).

For both agonist and antagonist activity, the G substituent, is an iminoacid, preferably Pro or Hyp; or an aliphatic amino acid, as describedabove for substituent B.

For both agonist and antagonist activity, the X substituent is GlyNH₂,AlaNH₂, aminoethyl (-NHCH₂ CH₃), aminopropyl [--NH(CH₂)₂ CH₃ ], oraminohydroxyethyl [--NH(CH₂)₂ OH]. Thus, the X substituent is an amideend group which may be an amino acid, in which case the novel peptide isa decapeptide, or a fragment, in which case the novel peptide is anonapeptide.

For both agonist and antagonist activity, substituent Y is hydrogen orC₁₋₄ alkyl, preferably isopropylmethyl.

The novel peptides of the present invention have as an essential featurea lactam dipeptide conformation constraining bridge between the α-carbonof Gly⁶ and the amino group of Leu⁷ of the LHRH peptide. This lactambridge is further illustrated in the following partial formulas fromLHRH and a novel peptide of the present invention: ##STR4##

In accordance with the present invention there is provided a method ofpromoting fertility, comprising administering to a patient in need ofsuch treatment, a therapeutically effective amount of a peptide of theformula: ##STR5## wherein: A is pyroGlu;

B is His;

C is aromatic amino acid, preferably a member selected from the groupconsisting of Phe, Trp, Tyr, and His;

D is Ser, Thr, or Ala;

E is aromatic amino acid, preferably a member selected from the groupconsisting of Phe, Trp, Tyr, and His;

F is amino acid with a basic side chain, preferably a member selectedfrom the group consisting of Lys, Arg, and Orn;

G is imino acid, preferably a member selected from the group consistingof Pro and Hyp, or aliphatic amino acid, preferably a member selectedfrom the group consisting of Ala, Leu, Ile, and Val;

X is GlyNH₂, AlaNH₂, aminoethyl, aminopropyl, or aminohydroxyethyl;

Y is hydrogen or C₁₋₄ alkyl, preferably isopropylmethyl; and

n is 2 or 3.

Dosage levels of the order of 60 μg. to 35 mg. per day are useful in thetreatment of the above-indicated conditions. For example, fertility ispromoted by the administration of from about 1 μg. to 0.5 milligrams ofthe peptide per kilogram of body weight per day. Advantageously, fromabout 1 μg. to about 300 μg. per kilogram of body weight, and especiallyfrom about 2 μg. to about 100 μg./kg. per daily dosage produces highlyeffective results.

For convenience, the novel peptides of the present invention have beendivided into LHRH agonist and antagonist peptides with respect to theiractivity in controlling fertility, the agonist peptides promotingfertility and the antagonist peptides reducing fertility. In fact,however, while the antagonist peptides will only reduce fertility, thefertility controlling activity of the agonist peptides is dosagedependent. As indicated above, dosage levels of the order of 60 μg. to35 mg. per day are useful in promoting fertility. On the other hand,dosage levels of the order of 0.5 mg. to 500 mg. per day are useful inreducing fertility. Thus, fertility is reduced by administration of fromabout 10 μg. to about 7.5 mg. of the peptide per Kilogram of body weightper day. Advantageously, from about 0.02 mg. to about 2 mg. per kilogramof body weight, and especially from about 0.03 mg. to about 1.0 mg./kgper daily dosage produces highly effective results.

In accordance with the present invention there is also provided a methodof reducing fertility comprising administering to a patient in need ofsuch treatment, a therapeutically effective amount of a peptide of theformula: ##STR6## wherein: A is L- or D- pyroGlu; L- or D- N-acetyl orN-pyroGlu imino acid, preferably a member selected from the groupconsisting of N-acetyl Pro, N-acetyl Hyp, N-pyro-Glu Pro, and N-pyroGluHyp; acetyl D-Phe; or C₃₋₇ cycloalkyl acyl, preferablycyclopropylcarbonyl;

B is Gly; L- or D- aromatic amino acid, preferably a member selectedfrom the group consisting of Phe, Trp, Tyr, and His; an L- or D-aliphatic amino acid, preferably a member selected from the groupconsisting of Ala, Leu, Ile, and Val; or is absent altogether;

C is L- or D- aromatic or aliphatic amino acid, as described above forsubstituent B;

D is Ser, Thr, or Ala;

E is aromatic amino acid, as described above for substituent B;

F is amino acid with a basic side chain, preferably a member selectedfrom the group consisting of Lys, Arg, and Orn;

G is imino acid, preferably a member selected from the group consistingof Pro and Hyp; or aliphatic amino acid, as described above forsubstituent B;

X is GlyNH₂, AlaNH₂, aminoethyl, aminopropyl, or aminohydroxyethyl;

Y is hydrogen or C₁₋₄ alkyl, preferably isopropylmethyl; and

n is 2 or 3.

Dosage levels of the order of 0.5 mg. to 500 mg. per day are useful inthe treatment of the above indicated conditions. For example, fertilityis reduced by the administration of from about 10 μg. to about 7.5 mg.of the peptide per kilogram of body weight per day. Advantageously, fromabout 0.02 mg. to about 2 mg. per kilogram of body weight and especiallyfrom about 0.03 mg. to about 1 mg./kg. per daily dosage produce highlyeffective results.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration of humans may containfrom 5 mg. to 1 gram of active agent compounded with an appropriate andconvenient amount of carrier material which may vary from about 5 toabout 95 percent of the total composition. Dosage unit forms willgenerally contain between from about 25 mg. to about 500 mg. of activeingredient.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, and drug combination.

In accordance with the present invention there is further providedpharmaceutical compositions for use in promoting fertility, and inreducing fertility. The novel peptides of the present invention possessa high degree of LHRH agonist and antagonist activity and arelong-acting.

For these purposes the novel peptides of the present invention may beadministered orally, topically, parenterally, by inhalation spray,intravaginally, or rectally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvantsand vehicles. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. In addition to the treatment of warm-bloodedanimals such as mice, rats, horses, dogs, cats, etc., the peptides ofthe present invention are effective in the treatment of humans.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide a pharmaceutically elegant andpalatable preparation. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for manufacture of tablets. These excipients may be, forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example maize starch, or alginic acid;binding agents, for example, starch, gelatine or acacia, and lubricatingagents, for example magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatealone or with a wax may be employed.

Formulations for oral use may also be presented as hard gelatinecapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatine capsules wherein the active ingredient is mixed withwater or an oil medium, for example arachis oil, peanut oil, liquidparaffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethyl-cellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a natural-occurring phosphatide, forexample, lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol mono-oleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidanhydrides and hexitol, for example polyoxyethylene sorbitan monooleate.The said aqueous suspensions may also contain one or more preservatives,for example, ethyl or n-propyl p-hydroxy benzoate, one or more coloringagents, one or more flavoring agents and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilsuspension may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents, such as those set forthabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavouring and colouringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oils, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifyling agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soya bean lecithin, andesters of partial esters derived from fatty acid anhydrides and hexitol,for example sorbitan mono-oleate, and condensation products of the saidpartial esters with ethylene oxide, for example polyoxyethylene sorbitanmono-oleate. The emulsions may also contain sweetening and flavouringagents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative and flavouring and coloring agents. Thepharmaceutical compositions may be in the form of a sterile injectablepreparation, for example as a sterile injectable aqueous or oleagenoussuspension. This suspension may be formulated according to the known artusing those suitable dispersing or wetting agents and suspending agentswhich have been mentioned above. The sterile injectable preparation mayalso be a sterile injectable solution or suspension in a non-toxidparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectibles.

The peptides of the present invention may also be administered in theform of suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials are coca butter and polyethyleneglycols.

The novel peptides of the present invention, with respect to the variousamino acid substituents of the peptide, are prepared in accordance withwell-known methods in the art for the synthesis of peptides. However,the unique lactam dipeptide bridge in the novel peptides of the presentinvention is prepared in accordance with a novel process, with which,accordingly, the present invention is also concerned. The novelpreparation method of the present invention may be illustrated by thefollowing diagram: ##STR7## R₁ is a C₁₋₄ alkyl group, preferably methyl;R₂ is the same as Y, defined above; and R₃ is C₁₋₁₂ alkyl, for examplet-butyl, C₁₋₄ ar or alkyl, for example, benzyl. The group is aprotective group, and may be, for example butyloxycarbonyl (BOC).

An additional feature, and unexpected advantage of the novel process ofthe present invention is conversion of most of the alkyl ester (COOR₁)to the carboxylic acid during the course of the reaction without therequirement of any additional treatment step. Separation of the acidproduct or, alternatively, hydrolysis of the small amount of esterproduct to acid may be carried out.

In the first step of the preparation method, there may be used as thestarting material, for example, the methyl ester of BOC protecteddipeptide Met-Leu (Methionine-Leucine). This starting material wouldthus be the compound of Formula IV. where R₁ is methyl, R₂ isisopropylmethyl, and R₃ is t-butyl.

The starting material is treated with an alkylating agent, for exampledimethylsulfate, diethylsulfate, methylfluorosulfonate, methyltrifluoromethanesulfonate, trimethyloxonium fluoroborate, preferablymethyl iodide, which, in the case of methyl iodide, may also serve asthe solvent medium for the reaction. However, any suitable anhydrousaprotic solvent can be used. A temperature of from 0° to 40° C. may beemployed. Preferably, the starting material is treated with methyliodide by admixing and stirring at room temperature for about two days.The resulting product is the sulfonium salt of the alkyl ester ofprotected-Met-Leu, illustrated by Formula V.

In the second step of the preparation method of the present invention,the sulfonium salt of Formula V. is treated with a strong base in orderto effect ring closure. The strong base may be an alkali metal hydride,preferably sodium hydride, but may also be, for example, lithiumdiisopropylamide or potassium t-butoxide. The reaction is carried out inan anhydrous aprotic solvent, for example methylene chloride anddimethylformamide (DMF), either separately or combined; alkyl ethers,for example dimethoxyethane; or tetrahydrofuran. The reactiontemperature is preferably about 0° C., but temperatures of -50° C. to50° C. are suitable.

The resulting product is a protected 2-(3-amino-2-oxypyrrolidin-1-yl)acetic acid which may be 2-alkyl substituted. This product isillustrated by Formula VI. and is a novel compound, as is thecorresponding unprotected compound, which may be prepared by removingthe protecting group through cleavage, for example by treatment with astrong acid or catalytic hydrogenation. Consequently, the presentinvention is also concerned with the following novel compounds: ##STR8##where R₂ is C₁₋₄ alkyl and R₃ is C₁₋₁₂ alkyl or C₁₋₄ aralkyl.

Thus, the novel method of preparation of the present invention is amethod of preparing a compound of the formula: ##STR9## where A ishydrogen or ##STR10## comprising the steps of

(1) treating a ##STR11## protected Met-aliphatic amino acid ester of theformula: ##STR12## with an alkylating agent, whereby the correspondingsulfonium salt is produced; and

(2) treating the sulfonium salt prepared in Step (1) with a strong basein order to effect ring closure and prepare the compound of FormulaVIII. where A is ##STR13##

(3) cleaving the ##STR14## protecting group from the compound of FormulaVI. to prepare the compound of formula VIII. where A is hydrogen.

The following example illustrates preparation of a novel peptide of thepresent invention.

EXAMPLEPyroGlu-His-Trp-Ser-Tyr-6,7-[2-(S-3-Amino-2-Oxo-Pyrrolidin-1-yl)-S-2-IsopropylmethylaceticAcid]-Arg-Pro-GlyNH₂

A. BOC-L-Met-L-Leu-OMe

6.225 g. (25 mole) of BOC-L-Met was dissolved in 160 ml. of degasseddimethylformamide and cooled under nitrogen to 0° C. 4.55 g. (25 mmole)of L-Leu-OMe was dissolved in 80 ml. of dimethylformamide and set aside.To the BOC-L-Met was added 5.38 ml. of diphenylphosphorylazide in 10%excess followed by 3.48 ml. of triethylamine in 10% excess, followed bythe L-Leu-OMe. The reaction mixture was stirred at 0° C. for 3 hrs.,then at room temperature for 16 hrs. The solution was concentrated andtreated with 3:1 dimethylformamide/water and mixed bed resin, whereuponit crystallized out on the resin. The resin and crystals were filteredand the resin washed with dimethylformamide to isolate the product. Thefiltrate was then concentrated and recrystallized from ethylacetate/hexane. 7.74 g. of product was obtained.

B. Sulfonium Salt of BOC-L-Met-L-Leu-OMe

7 g. (0.019 mole) of the ester product of A above was dissolved in 40ml. of neat methyl iodide and stirred at room temperature for 1 hr. Thereaction mixture was concentrated by water aspiration and in vacuodrying, and the residue was washed by evaporating with dichloromethaneand methanol to form a foam in 90% yield (9.13 g.).

C. Cyclization to formBOC-[2-(S-3-Amino-2-Oxo-Pyrrolidin-1-yl)-S-2-Isopropylmethylacetic Acid]

The sulfonium salt product of B. above (9.13 g.) was dissolved in 300ml. of a 1:1 mixture of dimethylformamide and dichloromethane and cooledto 0° C., under nitrogen. Two equivalents (1.5 g.) of a 50% dispersionof sodium hydride in mineral oil was added in one portion. After 2 hrs.100 ml. of methyl acetate was added and this was followed by 2 ml. ofwater to quench the reaction, after which the reaction mixture wasallowed to stand overnight. The reaction mixture was partitioned betweendichloromethane and water, and the water was acidified topH 4 withconcentrated citric acid and extracted three times with dichloromethane.The dichloromethane portion was dried over sodium sulfate andconcentrated to a crystalline mass. Two crops of crystals totaled 2.214g. The product is BOC-blocked2-(3-amino-2-oxo-pyrrolindin-1-yl)-2-isopropylmethyl acetic acid.

D. BOC-[2-(S-3-Amino-2-Oxo-Pyrrolidin-1-yl)-S-2-IsopropylmethylaceticAcid]-Arg-Pro-GlyNH₂

1.6 g. (5 mmole) of the2-(3-amino-2-oxo-pyrrolidin-1-yl)-2-isopropylmethyl acetic acid preparedin C. above was dissolved in 35 ml. of degassed dimethylformamide andcooled to 0° C. under nitrogen. 9.5 mmole of the TFA (trifluoroaceticacid) salt of H-Arg-Pro-GlyNH₂, previously prepared by method known inthe art, was dissolved in 15 ml. of degassed dimethylformamide, cooled,and set aside. To the acid solution was added 1.097 ml. (5.5 mmole) ofdiphenylphosphorylazide and 0.697 ml. (5.5 mmole) of triethylamine,followed by the pre-cooled peptide solution. The reaction mixture wasstirred at 0° C. for 3 hrs., then at room temperature overnight. Productwork-up involved a silica gel filtration with 70:30:3chloroform/methanol/aqueous ammonia. A foam product (2.5 g.) wasobtained.

E. Deblocking ofBOC-[2-(S-3-Amino-2-Oxo-Pyrrolidin-1-yl)-S-2-IsopropylmethylaceticAcid]-Arg-Pro-GlyNH₂

The peptide product of D. above was used as a foam and dissolved in 30ml. of trifluoroacetic acid containing 1% ethanedithiol (EDT) at 0° C.,after which it was stirred at room temperature for 15 min., followed bysolvent removal. Pumping and washing with ether removed most of the EDT.A foam product (2.77 g.) was obtained.

F.PyroGlu-His-Trp-Ser-Tyr-6,7-[2-(S-3-Amino-2-oxo-Pyrrolidin-1-yl)-S-2-IsopropylmethylaceticAcid]-Arg-Pro-GlyNH₂

950 mg. (1.3 mmole) of the hydrazide PyroGlu-His-Trp-Ser-Tyr-NHNH₂,previously prepared by methods known in the art, was dissolved in 13 ml.of degassed dimethylformamide and cooled to -10° C. under nitrogen in a1:1 methanol/water dry ice bath. There was then added 5 equivalents (6.5mmole) of 5.8 M hydrochloric acid/tetrahydrofuran (1.2 ml.). Thereaction mixture was cooled to -25° C. and there was added a 1:19solution of isoamylnitrite/dimethylformamide until a positive starch/KItest reaction was obtained. About 8 ml. of solution was required. Thinlayer chromatography showed no hydrazide remained. The reaction mixturewas cooled to -40° C. and the peptide product of E. above, cooled anddissolved in 2 ml. of dimethylformamide, was added. The pH was raised to8 with triethylamine. The reaction mixture was stored at -20° C. for 24hrs., after which the pH was readjusted. Additional peptide was added,but after 24 hrs. there wasno change in the thin layer chromatography ofthe product. The product was concentrated in vacuo, dissolved inbutanol, and partitioned with water. The butanol layer was placed on asilica gel column and eluted with 10:5:1:3 ethyl acetate/pyridine/aceticacid/water. The fractions were collected and concentrated, thenprecipitated with chloroform/hexane to give 150 mg. of final product.

What is claimed is:
 1. A novel peptide of the formula: ##STR15## whereinfor A the L- or D- N-acetyl or N-pyroGlu imino acid is a member selectedfrom the group consisting of N-acetyl-Pro, N-acetyl-Hyp, N-pyroGlu-Pro,and N-pyroGlu-Hyp; for B the L- or D-aromatic amino acid is a memberselected from the group consisting of Phe, Trp, Tyr, and His, and the L-or D- aliphatic amino acid is a member selected from the groupconsisting of Ala, Leu, Ile, and Val; for C the L- or D- aromatic aminoacid is a member selected from the group consisting of Phe, Trp, Tyr,and His, and the L- or D- aliphatic amino acid is a member selected fromthe group consisting of Ala, Leu, Ile, and Val; for E the aromatic aminoacid is a member selected from the group consisting of Phe, Trp, Tyr,and His; for F the amino acid with a basic side chain is a memberselected from the group consisting of Lys, Arg, and Orn; and for G theimino acid is a member selected from the group consisting of Pro andHyp, and the aliphatic amino acid is a member selected from the groupconsisting of Ala, Leu, Ile, and Val.
 2. A compound according to claim 1wherein A is pyroGlu; B is His, C is Trp; D is Ser; E is Tyr; F is Arg;G is Pro; X is GlyNH₂, Y is isopropylmethyl; and n is
 2. 3. A compoundaccording to claim 1 wherein A is pyroGlu; B is His; C is Trp; D is Ser;E is Tyr; F is Arg; G is Pro; X is aminoethyl; Y is isopropylmethyl; andn is
 2. 4. A compound according to claim 1 wherein A is pyroGlu; B isD-Phe; C is Trp; D is Ser; E is Tyr; F is Arg; G is Pro; X is GlyNH₂ ; Yis isopropylmethyl; and n is
 2. 5. A compound according to claim 1wherein A is N-acetyl-Pro; B is D-Phe; C is D-Trp; D is Ser; E is Tyr; Fis Arg; G is Pro; X is GlyNH₂ ; Y is isopropylmethyl; and n is
 2. 6. Anovel peptide of the formula: ##STR16## wherein: A is L- or D- pyroGlu;L- or D-N-acetyl- or N-pyroGlu-Pro; acetyl D-Phe; or C₃₋₇ cycloalkylacyl;B is His; Gly; L- or D-: Phe, Tyr, Ala, Leu, Ile or Val; or isabsent; C is L- or D-: Phe, Tyr, Ala, Leu, Ile, or Val; D is Ser; Thr;or Ala; E is Phe; or Tyr; F is Lys; or Arg; G is Pro; Ala; Leu; Ile; orVal; X is GlyNH₂ ; AlaNH₂ ; aminoethyl; aminopropyl; oraminohydroxyethyl; Y is hydrogen; or C₁₋₄ alkyl; and n is 2 or 3.